The last step of the ethylene biosynthesis pathway in turnip tops (Brassica rapa) seeds: Alterations related to development and germination and its inhibition during desiccation

The involvement of ethylene in zygotic embryogenesis is a little known aspect of the growth and development in higher plants. In the present work, we study the alterations of the last step of the ethylene biosynthesis pathway during the formation period of turnip tops ( Brassica rapa cv. Rapa) seeds and its repercussions in the germination process and post-germinative growth. For this, we chose 11 different phases of silique development, the first being the recently fertilized pistil and the last being the silique just prior to its dehiscence (ca. 2 months post-anthesis). In the 11 phases, ethylene production was detected in both whole silique (with or without seeds) and in the seeds enclosed by the silique wall. The levels of ACC, ACO and ethylene production proved high in seeds belonging to: (1) the pod in the very early phases, when the seeds were growing but without photosynthetic competence; (2) the silique at maximum growth, in which the seeds will initiate desiccation and loss of photosynthetic activity. During the phases prior to dehiscence, there was a marked inhibition in the last step of the ethylene biosynthesis pathway. In viable dry seeds, no ACO activity was detected and the ACC levels were 4-fold lower than at the onset of the silique senescence. Germination brings about a net synthesis of ACC with respect of the stores dry seed. This fact, together with other results presented in this work, point towards, as in other seeds, a dependence of ethylene synthesis for radicle emergence. The possible role played by the silique wall in the control of ethylene biosynthesis during zygotic embryogenesis, as well as the participation of ethylene as a hormonal signal in the triggering of seed desiccation in Brassica rapa cv. Rapa, are discussed in depth.     

Chlorophyllase and peroxidase activity during degreening of maturing canola (Brassica napus) and mustard (Brassica juncea) seed

Chlorophyllase and peroxidase activities were measured in relation to seed maturation and degreening in canola ( Brassica napus cvs Westar and Alto) and mustard ( Brassica juncea cvs Cutlass and Lethbridge 22A). Samples of seed collected at the same moisture content were pooled, then divided and used for each assay. During maturation the green pigment (chlorophyll and related pigments) content of canola seed decreased linearly and was lower than that measured in mustard at all moisture contents studied, except for the highest and lowest moisture contents. Chlorophyllides and pheophorbides were detected in canola and were essentially absent in mustard. This difference in accumulation of dephytylated pigments infers differences in the pigment degradation pathways in Brassica species. Interspecific differences in the enzymology of degreening were found. Green pigment degradation was associated with increased chlorophyllase activity and low peroxidase activity in canola and low Chlorophyllase and high perosidase activity in mustard. The possible role of ethylene in seed degreening is discussed.     

Relationship of chlorophyll, seed moisture and ABA levels in the maturing Brassica napus seed and effect of a mild freezing stress

Chlorophyll (Chl) retention by mature seed of canola as the result of an early frost or other environmental factors (the "green seed problem") causes serious economic losses. The relationship of seed degreening to rate of moisture loss by seed and silique and the role of ABA in this process were investigated as a function of developmental age. During the normal predesiccation stage (28–45 days after pollination), seed of Brassica napus (cv. Westar) loses Chl rapidly but seed moisture slowly. After a mild freezing stress, there is a rapid loss of moisture from silique walls, followed by accelerated loss of seed moisture. Chl degradation ceases at 35–45% seed moisture. ABA levels in silique walls of frozen plants (determined by enzyme‐linked immunosorbant assay) increased after freezing, apparently in response to moisture loss. In contrast, ABA levels in the seed increased dramatically 1 day after freezing, then decreased to control levels. The influence of the rate of seed moisture loss on Chl degradation was investigated by fast and slow drying of isolated seed under controlled humidity conditions. Seed dried rapidly at 22% RH retained most of its Chl, whereas seed dried slowly at 86% RH lost Chl as fast or faster than seed on control (unfrozen) plants. In all treatments, Chl loss stopped at about 40% seed moisture.     

Yield reduction in Brassica napus, B. rapa, B. juncea, and Sinapis alba caused by flea beetle (Phyllotreta cruciferae (Goeze) (Coleoptera: Chrysomelidae)) infestation in northern Idaho

Phyllotreta cruciferae is an important insect pest of spring-planted Brassica crops, especially during the seedling stage. To determine the effect of early season P. cruciferae infestation on seed yield, 10 genotypes from each of two canola species (Brassica napus L. and Brassica rapa L.) and two mustard species (Brassica juncea L. and Sinapis alba L.) were grown in 2 yr under three different P. cruciferae treatments: (1) no insecticide control; (2) foliar applications of endosulfan; and (3) carbofuran with seed at planting plus foliar application of carbaryl. Averaged over 10 genotypes, B. rapa showed most visible P. cruciferae injury and showed greatest yield reduction without insecticide application. Mustard species (S. alba and B. juncea) showed least visible injury and higher yield without insecticide compared with canola species (B. napus and B. rapa). Indeed, average seed yield of S. alba without insecticide was higher than either B. napus or B. rapa with most effective P. cruciferae control. Significant variation occurred within each species. A number of lines from B. napus, B. juncea, anid S. alba showed less feeding injury and yield reduction as a result of P. cruciferae infestation compared with other lines from the same species examined, thus having potential genetic background for developing resistant cultivars.     

Carbohydrate Content and Enzyme Metabolism in Developing Canola Siliques

Little biochemical information is available on carbohydrate metabolism in developing canola (Brassica napus L.) silique (pod) wall and seed tissues. This research examines the carbohydrate contents and sucrose (Suc) metabolic enzyme activities in different aged silique wall and seed tissues during oil filling. The silique wall partitioned photosynthate into Suc over starch and predominantly accumulated hexose. The silique wall hexose content and soluble acid invertase activity rapidly fell as embryos progressed from the early- to late-cotyledon developmental stages. A similar trend was not evident for alkaline invertase, Suc synthase (SuSy), and Suc-phosphate synthase. Silique wall SuSy activities were much higher than source leaves at all times and may serve to supply the substrate for secondary cell wall thickening. In young seeds starch was the predominant accumulated carbohydrate over the sampled developmental range. Seed hexose levels dropped as embryos developed from the early- to midcotyledon stage. Hexose and starch were localized to the testa or liquid endosperm, whereas Suc was evenly distributed among seed components. With the switch to oil accumulation, seed SuSy activity increased by 3.6-fold and soluble acid invertase activity decreased by 76%. These data provide valuable baseline knowledge for the genetic manipulation of canola seed carbon partitioning.     

Dynamic Metabolic Profiles and Tissue-Specific Source Effects on the Metabolome of Developing Seeds of Brassica napus

Canola (Brassica napus) is one of several important oil-producing crops, and the physiological processes, enzymes, and genes involved in oil synthesis in canola seeds have been well characterized. However, relatively little is known about the dynamic metabolic changes that occur during oil accumulation in seeds, as well as the mechanistic origins of metabolic changes. To explore the metabolic changes that occur during oil accumulation, we isolated metabolites from both seed and silique wall and identified and characterized them by using gas chromatography coupled with mass spectrometry (GC-MS). The results showed that a total of 443 metabolites were identified from four developmental stages. Dozens of these metabolites were differentially expressed during seed ripening, including 20 known to be involved in seed development. To investigate the contribution of tissue-specific carbon sources to the biosynthesis of these metabolites, we examined the metabolic changes of silique walls and seeds under three treatments: leaf-detachment (Ld), phloem-peeling (Pe), and selective silique darkening (Sd). Our study demonstrated that the oil content was independent of leaf photosynthesis and phloem transport during oil accumulation, but required the metabolic influx from the silique wall. Notably, Sd treatment resulted in seed senescence, which eventually led to a severe reduction of the oil content. Sd treatment also caused a significant accumulation of fatty acids (FA), organic acids and amino acids. Furthermore, an unexpected accumulation of sugar derivatives and organic acid was observed in the Pe- and Sd-treated seeds. Consistent with this, the expression of a subset of genes involved in FA metabolism, sugar and oil storage was significantly altered in Pe and Sd treated seeds. Taken together, our studies suggest the metabolite profiles of canola seeds dynamically varied during the course of oil accumulation, which may provide a new insight into the mechanisms of the oil accumulation at the metabolite level.     

The zygotic embryogenesis and ripening of Brassica rapa seeds provokes important alterations in the levels of free and conjugated abscisic acid and polyamines

Prior to the embryogenic study of Brassica rapa (turnip tops), the PA (polyamine) changes during flower development were determined. Whereas the flower was closed, no notable changes were found. However, pollination strongly stimulated the synthesis of the F (free) and ASC (acid soluble conjugated) forms, the pistil and not the petals being the main responsible. During embryogenesis, major alterations appeared in the synthesis of free and conjugated PA and ABA. Seeds, and not the silique wall, contributed most to the total content of the different F-PA (free polyamines) in the silique, this contribution involving all the PA studied. During seed desiccation, F-Put remained almost undetectable whereas F-Spd and F-Spm were very high. This is also applicable to ASC-Spd and ASC-Spm, this not being noted in the silique wall. During the first 5 stages of silique embryogenesis, the wall had higher levels of AIC-PA (acid insoluble conjugated polyamines) than in the seeds, primarily due to AIC-Spd and AIC-Spm. By contrast, during the middle and final phases AIC-PA was found more in the seeds than in the silique wall. The free and conjugated ABA showed the presence of 3 and 2 peaks in the seeds and the silique wall, respectively. The first corresponded to the onset of development, the second appeared when the highest moisture content was reached, and the third coincided with the triggering of carotenoid synthesis when the seed and the silique wall had lost more than 70–75% of their moisture. Finally, it is shown that the dry seeds of turnip tops were highly heterogeneous, not only in their germinative capacity, but also in the levels of free and conjugated ABA, which increased inversely to the germinative capacity.     

The role of chlorophyllase in degreening canola (Brassica napus) seeds and its activation by sublethal freezing

Chlorophyllase mediates dephytylation of chlorophylls and pheophytins during seed degreening in canola ( Brassica napus cv. Westar). Degreening can be correlated with chlorophyllase activity in vitro, but it is difficult to demonstrate in vivo activity because of low levels of the dephytylated breakdown products during rapid degreening. If, however, degreening is inhibited by sublethal freezing, chlorophyllide and pheophorbide accumulation can be related to the action of chlorophyllase. Changes in the rate of in vitro dephytylation during degreening and the dramatic increase following freezing may indicate enzyme activation and de novo enzyme synthesis. Evidence from Western blots is presented in support of de novo synthesis. It is concluded that failure of the seed to degreen following sublethal freezing does not result from a reduction in chlorophyllase activity.     

Floral nectaries and nectar production in brown mustard (Brassica juncea) and white mustard (Sinapis alba) (Brassicaceae)

 The dynamics and abundance of nectar secretion as well as sugar productivity were studied in flowers of brown mustard (Brassica juncea) cv. Małopolska and white mustard (Sinapis alba) cv. Borowska. Moreover, floral nectaries were examined under LM and SEM. In both cultivars lateral and median pairs of nectaries secreted nectar. However, differences were found in morphology and activity of these pairs. The lateral nectaries produced more nectar than the median ones. Nectar secretion started at loose bud and peaked during anther dehiscence. Average amount of nectarsecreted by 100 flowers of cv. Małopolska and cv. Borowska were 119.9 mg and 134 mg. Mean concentration of nectar was 26.7% and 23.4%, respectively. One hundred flowers of cv. Małopolska and cv. Borowska secreted 28.4 mg and 24.9 mg of sugars in nectar. Estimated sugar productivity per 1 ha of crop was 65.5 kg and 71.2 kg, respectively. Received August 28, 2002; accepted November 2, 2002 Published online: June 2, 2003     

Effect of 17-DMC on Phytohormone Level and Growth of Rapeseed Plants (Brassica napus)

The effect of 17-dimethylmorpholinium chloride (17-DMC), a quaternary ammonium salt, on stem growth and productivity of spring rape (Brassica napus L., cv. Star), and on phytohormone content in stem tissues was studied. We found that the effects of 17-DMC on stem growth (inhibition of linear growth, development of anatomical structure and mechanical properties) were manifested as the changes in stem hormonal balance, particularly, in a decrease in the contents of GA, zeatin, and IAA and an increase in the ABA content. Seed weight increased due to the retarding effect of 17-DMC on stem growth and the promotion of silique and seed development.     

Non-lethal freezing effects on seed degreening in Brassica napus

The effects of a non-lethal freezing stress on chlorophyll content, moisture level and distribution, and abscisic acid (ABA) levels were examined in siliques and seeds of Brassica napus (canola). A non-lethal freezing stress resulted in the retention of chlorophyll in seed at harvest that was most pronounced for seeds 28, 32 and 36 days after flowering (DAF). This increase was primarily due to an increased retention of chlorophyll a relative to chlorophyll b. Chlorophyll retention in seeds exposed to a non-lethal freezing stress correlated with an increased ABA catabolism, as measured 1, 3 or 7 days after the stress treatment. Although the non-lethal freezing stress had no significant effect on moisture content in seeds of siliques stressed at 28–44 DAF, moisture distribution, as viewed by magnetic resonance imaging, showed an uneven drying of 32 and 40 DAF siliques after exposure to the non-lethal freezing stress. Moisture was initially lost more rapidly from the silique wall between seeds, than in control non-stressed siliques. Increased moisture loss was not due to structural changes in the vasculature of the silique/seed of stressed tissues. These results are consistent with the hypothesis that a non-lethal freezing stress-induced decrease in ABA level, during seed maturation, effects an inhibition of normal chlorophyll a catabolism resulting in mature but green B. napus seed.     

Canola (Brassica napus) germplasm shows variable allelopathic effects against annual ryegrass (Lolium rigidum)

Aims

The allelopathic activity of canola (Brassica napus) germplasm was investigated using the important Australian weed, annual ryegrass (Lolium rigidum) as the target species.

Methods

Three different canola plant densities (10, 20, and 30 seedlings/beaker) of each of 70 world-wide genotypes were tested in vitro in close proximity to annual ryegrass seedlings.

Results

The allelopathic activity of canola, as measured by reduction in annual ryegrass root and shoot growth, increased with canola crop seedling densities. Density did not consistently influence shoot length of annual ryegrass. Greater shoot length suppression was observed in genotype cv. Rivette and BLN3343CO0402. The Australian genotype cv. Av-opal and the breeding line Pak85388-502 suppressed root length of ryegrass more than other genotypes, even at low densities. At the lowest density, the least allelopathic genotypes were cv. Barossa and cv. Cescaljarni-repka, although they became more allelopathic at higher density. An overall inhibition index was calculated to rank each of the canola genotypes. There were significant differences between canola genotypes in their ability to inhibit root and shoot growth in ryegrass.

Conclusion

Considerable genetic variation exists among canola genotypes for their allelopathic effects on annual ryegrass. Further investigation is required to determine the allelopathic mechanisms, particularly to identify the responsible allelochemical(s) and the gene(s) controlling the trait. This research suggests that highly allelopathic canola genotypes can be potential for controlling weeds such as annual ryegrass in integrated weed management programs.     

Cloning and analysis of a cDNA encoding an endo-polygalacturonase expressed during the desiccation period of the silique-valves of turnip-tops (Brassica rapa L. cv. Rapa)

During zygotic embryogenesis of turnip-tops (Brassica rapa L. cv. Rapa), the polygalacturonase activity (PG; EC 3.2.1.15), measured as a decrease in viscosity of polygalacturonic acid, reached a high when the desiccation process in the seeded silique was triggered and the valves had lost more than 70-75% of their moisture (45-50 DPA). The PG activity was not detected in any phases of developing seeds. This work also characterizes a cDNA with an open reading frame of 1303 bp and that codes for a putative PG called BrPG1. This falls into the category of clade-B, which includes PG related to shattering and abscission processes. The deduced BrPG1 sequence predicted a 434-residue-long precursor protein (46.7kDa) with a transit peptide sequence 23 amino acids long. A molecular mass of 44.3 kDa was calculated for the mature form of BrPG1, which showed high sequence similarity to PGA1 (97%) of B. napus (X98373) and ADPG1 (87%) of Arabidopsis thaliana (AJ002532). All conserved amino acids at the catalytic site of PGs belonging to clade-B were preserved on BrPG1. This BrPG1 gene was specifically expressed in the silique valves of turnip-tops and was temporally expressed at the beginning of its desiccation.     

High Temperature Induced Male and Female Sterility in Canola (Brassica napus L.)

Male and female sterility was induced in canola or rapeseed(Brassica napus L. cv. Westar) flowers grown under high temperatureconditions (32°C/26°C; day/night). At maturity, themajority of flower buds remained closed but had protruding stigmas.The stamens were reduced in size and the anthers showed abnormalmicrosporogenesis. The gynoecia, although normal in appearance,did not set seed and ovule development was aberrant. Flowersof the ogu CMS line of B. napus also showed similar ovular abnormalitiesunder high temperatures and male fertility was not restoredin them. The observations presented suggest that high temperaturesin the field may adversely affect the yield of canola. Temperature, male and female sterility, rapeseed, canola, Brassica napus     

Storage oil breakdown during embryo development of Brassica napus (L.)

In this study it is shown that at least 10% of the major storage product of developing embryos of Brassica napus (L.), triacylglycerol, is lost during the desiccation phase of seed development. The metabolism of this lipid was studied by measurements of the fate of label from [1-(14)C]decanoate supplied to isolated embryos, and by measurements of the activities of enzymes of fatty acid catabolism. Measurements on desiccating embryos have been compared with those made on embryos during lipid accumulation and on germinating seedlings. Enzymes of beta-oxidation and the glyoxylate cycle, and phosphoenolpyruvate carboxykinase were present in embryos during oil accumulation, and increased in activity and abundance as the seeds matured and became desiccated. Although the activities were less than those measured during germination, they were at least comparable to the in vivo rate of fatty acid synthesis in the embryo during development. The pattern of labelling, following metabolism of decanoate by isolated embryos, indicated a much greater involvement of the glyoxylate cycle during desiccation than earlier in oil accumulation, and showed that much of the (14)C-label from decanoate was released as CO(2) at both stages. Sucrose was not a product of decanoate metabolism during embryo development, and therefore lipid degradation was not associated with net gluconeogenic activity. These observations are discussed in the context of seed development, oil yield, and the synthesis of novel fatty acids in plants.     

In vitro seed maturation in Brassica rapa L.: Relationship of silique atmosphere to storage reserve deposition

The Brassica rapa L. silique is a self-contained environment that maintains hypoxia around the developing seeds, and in which carbon dioxide accumulates to very high concentrations (>30,000 ppm). How the silique microenvironment modulates the composition and amount of storage reserves in the seeds is of interest because of the important agricultural role played by canola (B. rapa and Brassica napus) as an oilseed. Because of the small volume and dynamic nature of this microenvironment in Brassica, a standardized system was needed to study the environmental role played in storage reserve deposition. For this purpose we have developed a silique culture system that permits maturation of seed in vitro. Siliques excised from plants just 11 days after pollination complete the ripening of their seeds after 20 days of culture in light (200 μmol/m²/s) on MS medium containing 30 g/l sucrose, 0.25 mg/l BAP, and 0.025 mg/l NAA. Cytochemical localization and biochemical analyses revealed that storage reserves were affected by the in vitro maturation system. Although following a comparable ripening timeline to that occurring on the plant, and producing fully germinable seeds, in vitro maturation resulted in a 40% reduction in seed weight and the mature seeds contained decreased lipid, but increased protein, starch and soluble carbohydrates. To study the internal atmosphere surrounding the seeds, we developed a method to capture silique gases in helium with subsequent quantification of O₂ and CO₂ in the sample by gas chromatography. Analysis of the internal silique atmosphere showed that in vitro siliques provided seeds with a less oxygenated environment than they experience attached to the plant. Carbon dioxide concentrations remained high later into the maturation sequence in vitro than on the plant. When sampling gases from siliques attached to plants, we found multiple samples from the same plant resulted in higher variance than when only a single silique was sampled, suggesting that connection to the plant directly influences internal silique gases. Lower O₂ in the in vitro siliques was correlated with depressed lipid content in their mature seeds, supporting the conclusion that oxygen availability limits lipid accumulation. Previous studies showed how environmental factors influence Brassica embryos grown in tissue culture. These systems fail to preserve the component of metabolic regulation that is enforced by the silique wall tissues. Our in vitro maturation system provides a useful tool for specialized investigations since both the gaseous and hormonal environments can be readily manipulated.     

Methyl jasmonate elicits a differential antioxidant response in light- and dark-grown canola (Brassica napus) roots and shoots

Since jasmonates have been shown to mimic some of the plant'sresponses to stress, the effect of methyl jasmonate on antioxidant enzymes andcompounds was investigated in roots and shoots of light- and dark-grown canola(Brassica napus cv. Westar). The pattern of superoxidedismutase isoforms activity was also investigated. When enzyme activities werecalculated on a per gram of fresh weight basis, nearly all enzymes examinedshowed enhanced activity. However, when these activities were calculated basedon the amount of protein, methyl jasmonate induced an increase only insuperoxide dismutase activity in the roots of both light- and dark-grownseedlings. The ascorbate level was found to be higher in treated shoots,whereasthe glutathione level was found to be higher in treated roots. We conclude thatthe plant's antioxidant response to methyl jasmonate may be mainlydetermined by the type of tissue rather than by the light conditions. However,this last factor appeared to be involved in some antioxidant componentresponse,e.g. catalase activity and glutathione content.